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1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

An animal model of intervertebral disc degeneration induced surgically by ventral nuclear herniation in the rabbit produces morphologic changes of disc degeneration. Histologic characteristics and proteoglycan changes have been studied at various times after herniation. After injury, there was metaplasia into fibrocartilage originating from the cells along the margins of the annular wound, with proliferation of cells changing almost the entire disc space into fibrocartilage. A vertebral osteophyte occurred through an endochondral ossification sequence. Aggregating proteoglycans had two periods of repletion in the early course of degeneration. The water content of the disc was rapidly but only transiently restored in the first two days after herniation, whilst the changes in the total proteoglycan content of the disc paralleled these changes. Hyaluronic acid content decreased rapidly after herniation, but the size of the proteoglycan monomers did not change with degeneration. It is suggested that loss of confined fluid mechanics signals an abortive repair attempt rather than that of biochemical changes in proteoglycans initiate disc degeneration.

Analysis of the knee-joint cartilage of pigs at five ages (namely foetuses from the second half of pregnancy and animals 10 weeks, 25 weeks, 3 years and 5 years old) showed that the composition approached that of adult cartilage by 25 weeks of age, the most marked differences being between foetal and 10 week-old cartilage. Protein–polysaccharides were extracted sequentially, first by brief low-speed homogenization with iso-osmotic sodium acetate, then by two extractions with 2m-CaCl2 for 24h with gentle agitation interspersed with brief low-speed homogenization and agitation for another 24h. About half of the protein–polysaccharides were removed from foetal cartilage by the first extraction and the remainder by the second. The proportion in the first extract declined sharply with the age of the animal, but that in the first CaCl2 extract was similar at all ages other than 10 weeks. The amount left in the residue increased approximately with the collagen content from about one-fifth at 10 weeks of age to one-third in adult and old cartilage. The proportion of medium-sized protein–polysaccharides in the extracts changed little with age after birth, but the glucosamine content increased about fivefold and the protein content almost doubled between 10 weeks and 5 years of age. Other analytical values changed little. These results cannot be explained solely by changes in the proportion of ‘link-glycoprotein’ in the protein–polysaccharides. Since major changes in most parameters had taken place by 25 weeks of age, the first weeks after birth may be a critical period for cartilage development in the pig.

The addition of small amounts of hyaluronic acid (0.01–1.0%) to disaggregated cartilage proteoglyans produced a large increase in hydrodynamic size on gel chromatography. Re-chromatography of the excluded and retarded fractions showed that there was no equilibrium between them. The increase in hydrodynamic size, which was unaffected by the presence of EDTA, was maximal when the proportion of hyaluronic acid was rather less than 1% and decreased with higher proportions up to 50%. There was no such interaction when proteoglycans were mixed with other polyanions in comparable proportions. The interaction was accompanied by a large increase in viscosity which was eliminated in concentrated solutions of guanidine·HCl and at low pH. From the optimal proportions of proteoglycan and hyaluronic acid and estimates of their molecular weights, it was calculated that 10–30 proteoglycans were associated with each hyaluronic acid chain.

Proteoglycans in the matrix of adult cartilage are metabolised quite rapidly [ 1,2], nevertheless adult cartilage has a limited capacity for repair. Embryonic cartilage on the other hand, is able to replace the proteoglycans of the matrix within a few days after they have been largely depleted by the action of enzymes [3,4], which suggests that the macromolecules in the environment exert some influence over the synthetic or secretory functions of chondrocytes. The possible influence of a variety of macromolecules on the biosynthesis of sulphated glycosaminoglycans has therefore been examined using suspensions of nondividing adult chondrocytes, obtained from pig laryngeal and articular cartilage. Cartilage contains small amounts of hyaluronic acid [5], low concentrations of which have important biological effects [6]. The effect of low concentrations of hyaluronic acid on the incorporation of 35SOiinto material precipitable by cetylpyridinium chloride (CPC) is reported here.

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

Proteoglycan extracted from the Kurloff cells of the guinea-pig spleen and its corresponding glycosaminoglycan inhibits the migration of macrophages from capillary tubes in vitro, but the proteoglycan has no effect on lymphocyte transformation in vitro. A proteoglycan, chemically and spectrally similar to Kurloff cell proteoglycan, extracted from human spleen, also inhibits macrophage migration. In contrast, proteoglycan from cartilage and its corresponding glycosaminoglycan had no significant effect. The differences between Kurloff cell proteoglycan and migration inhibitory factor (MIF) are discussed.

1. Protein–polysaccharides of chondroitin 4-sulphate were extracted with neutral calcium chloride from pig laryngeal cartilage that was not completely homogenized. The protein–polysaccharides were purified by precipitation with 9-aminoacridine. On zone electrophoresis in compressed glass fibre at pH7·2 it was separated into two fractions, although two distinct zones were not obtained. These fractions, which had already been shown to differ in their antigenic determinants, also differed considerably in amino acid composition, total protein, hexose and glucosamine contents. 2. The fraction of higher mobility contained approx. 2% of protein and only traces of glucosamine. Serine and glycine accounted for over half the total amino acid residues, but aromatic, basic and sulphur-containing amino acids were not detected. The weight-average molecular weight, determined by sedimentation, was 230000. 3. Assuming that there was the same sequence of neutral sugars at the linkage points as in PP-L fraction (protein–polysaccharide light fraction), the approximate molar ratio of hexose to serine suggested that most of the serine residues were linked to chondroitin sulphate chains. Support for this was derived from the agreement between the weight-average molecular weight of the chondroitin sulphate–peptide after proteolysis, and the chain weight calculated from its serine content. The chain weight based on the serine content of the fraction of higher electrophoretic mobility was approximately similar. 4. In contrast, the fraction of lower electrophoretic mobility resembled PP-L fraction in its amino acid composition, protein and glucosamine contents. The presence of glucosamine, together with the higher hexose content, suggested that this fraction contained some keratan sulphate. 5. The relatively low molecular weight of the fraction of higher mobility enabled it to be extracted without complete disintegration of the cartilage. The unlikelihood of its being produced by autolytic enzymes is discussed.

1. Protein–polysaccharides from pig laryngeal cartilage extracted by two procedures described in the preceding paper (Tsiganos & Muir, 1969) were shown to consist of macromolecules of various sizes as assessed by gel filtration in 4% and 6% agarose. 2. A larger proportion of the smaller molecules was present in the preparation obtained by brief extraction in iso-osmotic sodium acetate (procedure I) than in that obtained by more prolonged extraction in 10% (w/v) calcium chloride (procedure II). 3. Two fractions were separated by gel filtration in 6% agarose and by electrophoresis in compressed glass fibre. These fractions differed in chemical composition and in antigenic determinants. The gel-retarded fraction R and that of higher electrophoretic mobility possessed the same single antigen, whereas the gel-excluded fraction E and the slower electrophoretic fraction contained all the antigens of the starting material including that of fraction R. 4. Five N-terminal amino acid residues were identified in preparation I and fraction E, only two of which were present in fraction R. 5. The relative proportions of gel-excluded and gel-retarded fractions did not change when solutions of high ionic strength, urea or guanidine hydrochloride were used for elution. 6. The differences in chemical and amino acid composition between fractions R and E showed that the latter was not a simple aggregate of the former. Fraction E contained more basic and aromatic amino acids, and some methionine and cystine; the last two were absent from fraction R. Hydroxyproline was not detected in either fraction. 7. The number of glycosidic linkages in both fractions was estimated by alkaline β-elimination. Appreciable amounts of threonine as well as serine were destroyed in both fractions. An average chain length for chondroitin sulphate was calculated from the galactosamine content of both fractions and the amounts of hydroxy amino acid destroyed. Average chain lengths were also calculated from the xylose and galactosamine content of each fraction. Each independent method gave a value of approximately 28 disaccharide units for the chain length in both fractions and hence their difference in size could not be explained by differences in the length of carbohydrate chains. 8. All fractions contained glucosamine, which was attributed to keratan sulphate. Content of both protein and keratan sulphate increased with the size of the macromolecules. 9. It is suggested, from these results, that chondroitin sulphate–protein complexes normally exist as a heterogeneous population of macromolecules in cartilage, and that keratan sulphate is involved in the formation of larger molecules.

Protein–polysaccharides of knee-joint cartilage of 9-month-old pigs were extracted sequentially with neutral iso-osmotic sodium acetate after five repeated homogenizations. One-third of the uronic acid originally present in the tissue was brought into solution, about half being in the first extract. The protein–polysaccharides, which were purified by precipitation with 9-aminoacridine, were heterogeneous in size on gel chromatography. The smallest (retarded by 6% agarose) were the most easily extracted since they were most prevalent in the initial extracts and absent from later ones, whereas the proportion of larger molecules increased progressively in successive extracts. Nevertheless a small proportion of the largest molecules (excluded from Sepharose 2B) was present even in the first extract. None of the protein–polysaccharide preparations contained hydroxyproline, and the analyses of their constituent sugars were the same, although there was a progressive increase in the protein content and in the glucosamine/galactosamine molar ratio of successive extracts. In each preparation this molar ratio was invariably greater in larger than in smaller molecules separated by gel filtration. From galactosamine/pentose molar ratios it appeared that the chondroitin sulphate chains were on average about 29 disaccharide units in length in the protein–polysaccharides of each extract, although gel-chromatography and cetylpyridinium chloride elution profiles showed that a somewhat higher proportion of shorter chondroitin sulphate chains occurred in the larger protein–polysaccharides. In the last extract, where the largest molecules predominated, about half could be reversibly dissociated by urea, whereas this had no effect on the protein–polysaccharides of earlier extracts even though these contained some large molecules.

1. Mucopolysaccharides were analysed in the urine of thirteen patients with Morquio's syndrome aged between three and fifty-nine years and of fourteen controls of comparable ages. 2. There were no significant qualitative or quantitative differences between patients and controls. 3. The clinical and radiological findings suggested that these patients did not have the "Morquio-Ullrich" form of the disease, which appears on retrospective assessment of case reports to be more uniform and less diffuse than the Morquio-Brailsford form which may include a number of possibly unrelated diseases. 4. Keratosulphate has so far been demonstrated in the urine only of patients with the "Morquio-Ullrich" form of the disease, although the mucopolysaccharide excretion has been investigated in only a few patients with the Morquio-Brailsford form. The normal mucopolysaccharide excretion of the present series of patients suggests that a normal mucopolysaccharide excretion distinguishes the Morquio-Brailsford from the Morquio-Ullrich form, the latter having a number of features overlapping with Hurler's disease, where large amounts of mucopolysaccharide other than keratosulphate are excreted. 5. Both qualitative and quantitative analysis of urinary mucopolysaccharide are thus necessary to distinguish between Hurler's disease, the Morquio-Ullrich form and the Morquio-Brailsford's form of Morquio's syndrome.

A method is described of measuring the tensile stiffness and fracture stress of human femoral condylar cartilage in planes parallel to and at increasing depth below the articular surface. The axis of tension was either parallel or perpendicular to the predominant collagen fibre direction in the superficial zone. Specimens were analysed for their collagen and glycosaminoglycan contents and partial correlation coefficients were determined between the tensile properties and each of the chemical constituents. The correlations between the tensile properties and the collagen content of specimens oriented parallel to the collagen fibre direction was statistically significant in the superficial zone but the significance level decreased with increasing depth. In specimens which were oriented perpendicularly to the collagen fibre direction the correlations between the above variables were less significant. There was no significant correlation between the tensile properties and the glycosaminoglycans in cartilage. Visibly normal specimens from the superficial layer which were situated adjacent to visibly degenerate cartilage were weaker and less stiff than specimens situated on normal joints or remote from visibly degenerate cartilage. Such differences decreased with depth below articular surface and were greater in parallel-oriented specimens.

1. (1) Conditions under which pentoses and methylpentoses react with anthrone in sulfuric acid have been reexamined. 2. (2) The number of steps involved has been reduced and good reproducibility obtained. 3. (3) Xylose was the most reactive pentose; other pentoses and methylpentoses required longer times of heating at 40°C. These differences in reaction rates and absorption spectra were utilized to estimate fucose and xylose in a mixture. 4. (4) Interference due to hexoses, hexuronic acids, hexosamine, sialic acid, and deoxyribose was virtually eliminated and no interference by tryptophan and protein was observed. 5. (5) Bound fucose and xylose reacted as easily as the free sugars. Although ribose in RNA did not, nevertheless the method could be used to estimate RNA in the presence of DNA provided an RNA standard is used.

Explants of cartilage from tibiae of 11–12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.

Pig articular cartilage, from which protein–polysaccharides soluble in iso-osmotic sodium acetate had been removed, was extracted in three further stages with 8m-urea in 2m-sodium acetate and with tris–HCl buffer after bacterial collagenase digestion, followed by the same urea–sodium acetate solution, thus leaving only 2% of the original uronic acid in the tissue. The histological appearance of the cartilage was unaltered until after collagenase digestion. The collagenase used did not affect the viscosity or molecular size of a protein–polysaccharide preparation obtained previously. The protein–polysaccharides in each extract differed in size, amino acid composition and protein content, but protein and keratan sulphate contents were not related to hydrodynamic size, in contrast with protein–polysaccharides extracted previously before collagenase digestion. Hydroxyproline could not be removed from those obtained by the first urea–sodium acetate extraction until degraded by heat. The galactosamine/pentose molar ratio agreed closely with the galactosamine/serine molar ratio that was destroyed on treatment with 0.5m-sodium hydroxide, showing that chondroitin sulphate was attached only to serine residues. From these molar ratios the chondroitin sulphate chains were calculated to be of the same average length in protein–polysaccharides in all three extracts although somewhat shorter than in protein–polysaccharides extracted previously. Some threonine residues were also destroyed on alkali treatment suggesting that keratan sulphate may be attached to threonine. These findings together with previous results show that differences in size, composition and physical state extend to all the protein–polysaccharides in cartilage.

SUPPLEMENTATION of deficient enzymes essential for complete catabolism of glycosaminoglycans (GAG) has been used with limited success in several types of mucopolysaccharidosis1–4. The beneficial effects and concomitant changes in urinary GAG after this form of treatment, however, have been only transient, presumably because of the short life in vivo of the enzymes involved5–7. Because of this limitation, we recently tried, by means of skin transplantation, to provide a more permanent source of corrective enzymes in a patient with Hunter syndrome8. Although two HLA antigens from each donor were incompatible with those of the patient and both grafts had been visibly rejected within 3 months, there was a marked increase in breakdown and excretion of GAG subsequent to treatment, which lasted for more than 9 months. In addition, the activity of Hunter corrective enzyme isolated from the patient's urine, was also significantly increased. We attributed the effectiveness of the skin transplant to the release of Hunter corrective factor by donor cells and its uptake by host cells, in a manner analogous to that described for fibroblasts in vitro9–11. We have now attempted to increase further both the effectiveness and longevity of replacement therapy, using fully histocompatible skin fibroblasts injected sub-cutaneously as a source of corrective enzyme. An advantage of this procedure is that surgery is not required and it would in principle be applicable to other genetic deficiency diseases of lysosomal enzymes.

Proteoglycans of cartilage when extracted by mild methods contain both aggregated and non-aggregated molecules (Sajdera & Hascall, 1969). In the presence of 4~-guanidinium chloride aggregates were dissociated and could be separated by equilibrium densitygradient centrifugation into a glycoprotein-like fraction containing little uronic acid and a proteoglycan fraction of lower protein content (Hascall & Sajdera, 1969). On mixing of both fractions in 4~-guanidinium chloride followed by dialysis to low ionic strength, proteoglycan aggregates were re-formed. When proteoglycans from pig laryngeal cartilage were fractionated in a similar way most of the proteoglycan separated from a glycoprotein-like fraction (Tsiganos et al., 1971), but when they were mixed the proteoglycans did not reaggregate (Tsiganos et a[., 1972). In contrast, the fraction from the middle of the density gradient, which contained only 5 % of the total uronic acid and 7.2 % of the protein, when mixed with the proteoglycan produced an increase in hydrodynamic size on gel chromatography, although there was no marked change in sedimentation rate in the ultracentrifuge (R. Pain, personal communication). The effect was thus similar to but not identical with the reaggregation of subunits (Hascall & Sajdera, 1969). The component responsible for the increase in hydrodynamic size of proteoglycans has now been purified and characterized. Proteoglycans were extracted from fresh pig laryngeal cartilage with 4~-guanidinium chloride in 50m~-sodium acetate buffer, pH4.5, and purified by equilibrium densitygradient centrifugation (Tsiganos et al., 1971). The proteoglycans were fractionated in a second CsCl gradient (po = 1.5) in 4~-guanidinium chloride, in an angle rotor (8 x 25ml) in an MSE65 centrifuge, at 95000g,,. for 48h at 20°C. Three fractions, obtained from each tube by freezing and cutting, corresponded to the bottom 4nd, middle lOml and top 4ml of the gradient. The distributions of total uronic acid and protein respectively were: bottom, 92.7 and 61 7;; middle, 5.0 and 7.2%; top, 2.3 and 31.8%. The ability of samples to produce an increase in hydrodynamic size of disaggregated proteoglycans was assessed as follows. Samples were added to proteoglycans in 4 ~ guanidinium chloride, dialysed against 0.5 M-sodium acetate buffer, pH 6.8, and chroniatographed in this buffer on a column (165cm x 1.1 cm) of Sepharose 2B. The uronic acid content (Bitter & Muir, 1962) of the eluate fractions was determined by an automated method (D. Heinegard, personal communication). Since little of the disaggregated proteoglycans was eluted in the void volume before the mixing, the extent of interaction was calculated from the proportion that did so after the mixing. When the bottom and middle fractions were mixed in the same relative proportions in which they occurred in the extract, the hydrodynamic size of about half the proteoglycans increased, whereas mixing of the top and bottom fractions did not produce this effect (Tsiganos et al., 1972). A sample of the middle fraction containing 0.93 mg of uronic acid was dialysed exhaustively against distilled water, conc. HCI was added to give a concentration of 20nm and the sample was applied to a column (1 6.0cm x 1.1 cm) of ECTEOLA-cellulose (Serva, Heidelberg, Germany) previously washed with 4M-HCI followed by distilled water until the eluate was pH5. The sample was washed in with 20ml of ~ O ~ M H C I and then eluted with 20ml each of 0 .5~-NacI , 2 .5~-NaCl and ~M-HCI. Acidic fractions were neutralized as soon as they cmerged from the column, and the last two fractions were dialysed against a large excess of 0.5 M-sodium acetate. Uronic acid contents of a11 fractions were determined, and their interactions with proteoglycan were tested. Fig. I shows the distribution of uronic acid of the fractions and the ability of each to interact with proteoglycans. Most of thc uronic acid (83 %)was eluted by 2 .5~-NaCl and ~ M H C I , whereas most of the capacity to interact witfi proteoglycan (73.2%) was eluted by 0.5 r~i-NaCl. This fraction contained equimolar amounts of hexuronic acid and hexosaniine, and the glucosaiiiiiie/galactosamine molar ratio was 25 : 1.

We acknowledge the excellent technical assistance of Miss Diane Orton and Mr. S. Brown, and thank the Medical Research Council for a grant to M. F. D., the Arthritis and Rheumatism Council, the Wellcome Trust and the Spastics Society for financial support. The clinical work described, for which the informed consent of the patients was obtained, was carried out with the full approval of the Ethical Committee of Guy's Hospital Medical School.

SUMMARY Infusion of a dextran solution of equivalent osmolality to plasma into a patient with the Sanfilippo syndrome caused an increased excretion of uronic acid. This increase was less than that obtained previously, when an equal amount of plasma was infused into the same patient. Moreover, there were no significant changes in the ratio of larger to smaller glycosaminoglycan fragments or in the sulphate/uronic acid ratios, in contrast to the changes noted subsequent to plasma infusion. It is concluded, therefore, that in this case infusion of dextran was not as effective as plasma in inducing a temporary increased mobilisation of pathological glycosaminoglycans. RESUME Effet de perfusion de dextrane sur l'excretion des glucosaminoglycuroniques dans le syndrome de Sanfilippo La perfusion de solution de dextrane isotonique au plasma chez un malade presentant un syndrome de Sanfilippo a provoque une excretion accrue d'acides uroniques. Cet accroissement a ete moindre que celui obtenu anterieurement, apres perfusion d'une quantiteequivalente de plasma chez le meme sujet. De plus, il n'y a pas eu de modifications significa-tives dans le rapport des fragments glucosaminoglycuroniques lourds et legers, ou dans le rapport sulfate-acide uronique, comme cela avait ete note anterieurement lors de la perfusion de plasma. Les auteurs concluent alors que, dans ce cas, la perfusion de dextrane n'a pas ete aussi efficace que celle de plasma pour provoquer un accroissement temporaire de mobilisation des glucosaminoglycuroniques pathologiques. ZUSAM MENFASSUNG Die Wirkung von Dextraninfusionen auf die Glycosaminoglycanausscheidung beim Sanfilippo-Syndrom Die Infusion einer Dextranlosung mit plasmaaquivalenter Osmolalitat steigert bei einem Patienten mit Sanfilippo-Syndrom die Ausscheidung von Harnsaure. Dieser Anstieg war geringer als er bisher durch Infusion gleicher Mengen Plasma bei demselben Patienten erreicht wurde. Daruberhinaus gab es keine signifikante Anderungder Rate vongroszlig;erenzu kleineren Glycosaminoglycanfragmenten oder im Sulfat/Harnsaureverhaltnis wie sie im Anschlus an Plasmainfusionen beobachtet wurden. Die Autoren stellen daher fest, das in diesem Fall die Infusion von Dextran nicht so wie die von Plasma die zeitweise verstarkt auftretende Mobilisation pathologischer Glycosaminoglycane bewirkte. RESUMEN Efecto de perfusiones de dextrano sobre la excrecion de glucosaminogliean en el sindrome de Sanfilippo La perfusion de una solution de dextrano de una osmolaridad equivalente a la del plasma a un paciente con sindrome de Sanfilippo causo una excrecion aumentada de acido uronico. Este aumento fue menor del obtenido previamente, al perfundir una cantidad igual de plasma en el mismo paciente. Ademas no hubo cambios significativos en la relacion entre los fragmentos mas grandes y mas pequenos de glycosaminoglican, o en la relacion sulfato/acido uronico, como los observados tras una perfusion de plasma. Los autores concluyen, por lo tanto, que en este caso la perfusion de dextrano no fue tan efectiva como la de plasma para inducir una movilizacion temporal aumentada de glycosaminoglicanos patologicos.

The glycosaminoglycans in the menisci of beagles 5--7 years old were analyzed at various times after osteoarthritis was induced by sectioning the anterior cruciate ligament of one knee; the unoperated knee served as control. In the first month after induction, there were signs of inflammation in the operated joint. After 1 week, the water content was elevated and the glycosaminoglycan content (per dry weight) was reduced. The content of keratan sulfate decreased more than that of chondroitin sulfate, but the hyaluronic acid content did not change consistently. The relative proportions of chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate remained unchanged. After 3--18 months, the glycosaminoglycan levels reverted to normal, and there was some evidence that after 15--18 months, they were elevated above normal. These results, together with results obtained from single examples of mild and severe osteoarthritis in working foxhounds, suggest that, in contrast to articular cartilage, the meniscus is capable of some regeneration in response to injury.

Electric birefringence measurements are reported for dilute aqueous solutions of proteoglycans. Complicated birefringence transients obtained over a range of applied electric fields of up to 6 kV cm-1 amplitude are shown to be consistent with the presence of three discrete relaxation phenomena, involving different rate processes. Analysis of the relaxation times suggests that self-association of the proteoglycan molecules occurs in the form of dimers which appear to be of head-to-head form. Single proteoglycan molecules and dimers co-exist in solution and contribute independent components to the birefringence at relatively low field strengths. With high applied fields, a third birefringence component can be observed which is attributed to motion of the chondroitin sulphate side chains in the field. In the presence of hyaluronic acid the proteoglycan molecules form complexes which are consistent with the model in which proteoglycans attach radially to extended hyaluronic acid chains. Proteoglycan samples for which the cystine residues have been reduced and alkylated show no evidence of this association with hyaluronic acid indicating the importance of the conformation of the proteoglycan molecule in its ability to bind to hyaluronic acid.

Osteoarthritis is a widespread disorder of vertebrate synovial joints in which progressive thinning of articular cartilage occurs at focal sites until in severely advanced stages, the underlying subchondral bone is exposed (Lee et al., 1974; Howell et al., 1976; Peyron, 1979). It is common in domestic animals, and in man the highest incidence is in late middle age. Osteoarthritis is diagnosed only at relatively advanced stages of the disease and it may be 20 years or more before clinical signs or radiological changes appear. There are probably a number of predisposing factors, including genetic predisposition but deterioration and abrasion of the articular surface strongly implicates mechanical factors. Formation of new bone at the articular surface and joint margins which accompanies the deterioration of cartilage suggests that the joint is an internally interacting system and that there is no single point in the pathological process (see Sokoloff, 1969). Because of its slow progress, osteoarthrosis is a difficult disease to study and hence its pathogenesis is poorly understood.

The molecular species of collagen in the human intervertebral disc have been identified and measured quantitatively by assay for specific peptides produced by digestion of whole tissue with cyanogen bromide. As previously found in the pig, the annulus fibrosus of lumbar intervertebral discs contained two genetically distinct molecular types of collagen, types I and II, whereas predominantly type II collagen was found in the nucleus pulposus. In each tissue the proportion of hydroxylysine residues in the total collagen that were glycosylated gave support to these findings, even though the contents of hydroxylysine alone provided an unreliable index of molecular type. The annulus fibrosus of human lumbar discs as a whole contained a considerably higher proportion of type II collagen (50–65% of total collagen) than did pig annulus fibrosus (20% of total collagen). No significant variations with age were found in the relative proportions of types I and II collagens in the annuli fibrosi from lumbar discs of individuals aged 5, 16, 59 and 66 years. At all ages more than 85% of the collagen in the nucleus pulposus was type II. In comparison, using the same procedure in the semi-lunar meniscus of the knee, which is classified as fibrocartilage, at least 95% of the collagen was type I, whereas more than 95% of that in young or old human articular cartilage was type II. The distributions of each type of collagen were assessed in a radial section of the annulus of T12/L1 of a 5 year old spine. Their relative proportions varied inversely and smoothly from being almost all type I collagen at the outer edge of the annulus to only type II on reaching the nucleus pulposus. This distribution already shown in pig annulus fibrosus appears to be a characteristic structural feature of this tissue.

The structure of the proteoglycans from normal pig nucleus pulposus and relatively normal human annulus fibrosus and nucleus pulposus was investigated in detail and the results were compared with the current structural model of proteoglycans of hyaline cartilage. Like proteoglycans of cartilage, those of intervertebral disc contain keratan sulphate and chondroitin sulphate attached to a protein core; they are able to aggregate to hyaluronic acid; the protein core likewise has three regions, one lacking glycosaminoglycans, another rich in keratan sulphate and a third region rich in chondroitin sulphate. However, disc proteoglycans contain more keratan sulphate and protein and less chondroitin sulphate and are also considerably smaller than cartilage proteoglycans. In proteoglycans of human discs, these differences appeared to be due principally to a shorter region of the core protein bearing the chondroitin sulphate chains, whereas in proteoglycans of pig discs their smaller size and relatively low uronic acid content were due to shorter chondroitin sulphate chains. There were subtle differences between proteoglycans from the nucleus and annulus of human discs. In the latter a higher proportion of proteoglycans was capable of binding to hyaluronate.

The findings establish that type III collagen is a major constituent of grossly proliferated rheumatoid and normal synovium. Unlike the collagen of normal synovium most of that in rheumatoid tissue could be solubilised by pepsin at 4 degrees C. Moore than half the pepsin-solubilised collage was identified as type III, the remainder being type I, by CM-cellulose chromatography; SDS-polyacrylamide electrophoresis with and without reduction of disulphide bonds; and amino acid analysis. Moreover, at least half the total collagen in several samples of normal as well as rheumatoid tissue was clearly type III when cyanogen bromide-derived peptides were run on SDS-polyacrylamide electrophoresis and compared with peptides prepared from purified types I and III collagens. This conclusion was supported by the isolation on phosphocellulose and quantitation by amino acid analysis of the collagen peptides alpha(1)CB2 and alpha(III)CB2 from a cyanogen bromide digest of rheumatoid synovium.

The mucopolysaccharidoses are a group of connective tissue disorders, classified by McKusick (1966), in which specific recessively inherited enzyme deficiencies result in an inadequate catabolism of glycosaminoglycans (GAGs), leading to an excessive accumulation within the tissues and a greatly increased urinary excretion. In pregnancies where it is suspected that the foetus may have inherited one of these disorders, amniocentesis is often carried out in order to ascertain the GAG composition of the amniotic fluid as a diagnostic aid to prophylactic abortion. A number of instances have now been reported (Matalon, Dorfman, Nadler and Jacobson, 1970; Matalon and Dorfman, 1972a; Crawfurd et al., 1973) in which qualitative and quantitative GAG abnormalities have been detected in the amniotic fluid of suspected cases, presumably due to excretion by the foetus. We have carried out further studies of foetal tissue from two cases of Hurler’s syndrome in which GAG abnormalities have been detected following amniocentesis and, in both cases, the GAG abnormalities in the amniotic fluids reflected the abnormalities in the foetal tissues.

Enzyme Replacement Therapy by Transplantation of HLA-Compatible Fibroblasts in Sanfilippo A Syndrome

The turnover of proteoglycans (assessed by the release into the medium of newly synthesised [35S]-proteoglycan) in explant cultures of articular cartilage from various anatomical sites of the knee joints (stifle) of mature beagles with experimental osteoarthritis has been studied with the following findings: (a) The proportion of newly synthesised proteoglycans released from cartilage explants maintained in vitro was generally increased for cartilage from operated compared with nonoperated control joints. (b) At 3 weeks after surgery there was a significant increase in the release of [35S]-proteoglycans from explants of the lateral and medial tibial plateaux of operated joints compared with sham-operated joints but not from other sites. On the other hand, when this comparison was made at 3 to 6 months after surgery, significant increases in the release of [35S]-proteoglycans were observed from cartilage of all anatomical areas except the patellar groove. (c) The release of [35S]-proteoglycan from cartilage explant cultures was dependent on live chondrocytes, since freeze-thawing the tissue immediately after labelling markedly reduced the release from both normal and osteoarthritic cartilage.

An animal model of intervertebral disc degeneration produced by surgical ventral disc herniation in the rabbit is described. Histologic studies showed proliferation of cells on the inner third of the annular wound, with metaplasia into fibrocartilage in the first 2 weeks following injury. Progressive fibrocartilaginous change occurred, reproducing the morphologic changes of disc degeneration over the first 6 weeks involving nearly the entire disc. Proteoglycans (total and newly synthesized) were studied qualitatively and quantitatively for periods of 1 to 200 days after herniation. There were two periods of time during the early course of degeneration when the ability of the proteoglycans to aggregate by interaction with hyaluronic acid was recovered, but this decreases progressively after 6-7 weeks. There was an immediate loss of water content from the injured disc which was restored only transiently during the first 2 days after herniation. Thereafter the water content progressively decreased. The uronic acid content of the disc changed in parallel with the changes in water content. The hyaluronic acid content decreased rapidly after herniation. However, the size of the proteoglycan monomers did not change with degeneration. The biochemical and morphologic changes are correlated, and an early repair mechanism is postulated to exist after injury.

We have developed techniques for the electrophoresis of 35S-labeled proteoglycans on polyacrylamide-agarose gel slabs and subsequent fixation, impregnation, and fluorography of such electrophoretograms. The procedure permits the examination of newly synthesized proteoglycan subspecies using a rapid technique, previously unavailable for these labeled molecules.

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 × 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 × 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol.wt. 670 000), an average Kd of 12 × 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

The biosynthesis of proteoglycans in the menisci and articular cartilages of the knee (stifle) of mature beagles was studied in the early stages of experimental osteoarthritis. The rate of proteoglycan synthesis, determined by systemic labeling in vivo at 21, 42, and 84 days after sectioning of the anterior cruciate ligament, was generally found to be 1.5-2.5-fold higher than control in articular cartilages and 3-10-fold higher than control in menisci. The medial meniscus was more stimulated than the lateral meniscus, and the medial tibial plateau under the meniscus was more stimulated than the adjacent tibial area. This area-specific stimulation suggests the involvement of mechanical factors in the cellular response. The rate of proteoglycan synthesis determined in vitro at 7, 14, and 21 days after operation was also about 2-fold higher than control in articular cartilages and about 3-fold higher in menisci. This increase in biosynthetic activity in vitro was confirmed by 35S-autoradiography and appeared to be due to general stimulation of existing chondrocytes, particularly in the middle and deep zones of the articular cartilage and throughout the meniscal cartilage. The rate of proteoglycan synthesis determined in vitro in cartilages from 2-week and 3-week sham operated joints was also increased relative to controls, suggesting that humoral as well as mechanical factors are involved in stimulating chondrocyte activity.

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μ m ) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.

Vitamin A toxicity has been associated with alterations in mineral metabolism and may result in osteopenia, fractures, deformities, and growth arrest. The pathogenesis of the bone lesions that occur in vitamin A toxicity is, however, ill defined and was examined in the present study. The administration of pharmacological doses of vitamin A to growing male rats resulted in weakness and spontaneous fractures. Undecalcified bone histology of vitamin A toxic animals was characterized by increased bone resorption, osteoclastosis, a paucity of trabecular surfaces covered with osteoid, and lesions which appear to be pathognomonic of hypervitaminosis A. The serum calcium and magnesium levels of vitamin A-toxic animals were unremarkable, but serum phosphate levels were significantly higher than control values. Urinary hydroxyproline excretion reflected bone histology and was significantly increased in experimental rats. Circulating levels of the potent bone resorbers, PTH, 1,25-dihydroxyvitamin D, and 25-hydroxyvitamin D, were, however, comparable in vitamin A-toxic and control animals, suggesting a possible direct effect of vitamin A on bone. Subsequently, the effects of vitamin A (retinol) on in vitro collagen synthesis (incorporation of [3H]proline into collagen) and bone resorption (45Ca release from bone) were examined using a fetal rat calvarial culture. Retinol added to the culture medium for 20-24 h in concentrations ranging from 0.5-10 micrograms/ml selectively inhibited collagen synthesis in a dose-dependent fashion. Higher concentrations of retinol were toxic and resulted in a general inhibition of protein synthesis. Bone resorption was stimulated by 0.5 and 2.5 micrograms/ml retinol. We conclude that vitamin A toxicity in rats causes bone lesions, the genesis of which can be explained, at least in part, by a direct effect of the vitamin on skeletal tissue.

ATTEMPTS to treat the mucopolysaccharidoses with infusions of normal plasma or leukocytes have produced mixed results; some workers1–3 reported little or no positive effect, while others4–6 noted both clinical and biochemical changes after treatment. Among the more obvious reasons for these conflicting findings are differences in the responsiveness to treatment of genetically distinct forms of the disorders7, in the volumes of plasma administered and in the activities of the corrective factors in the plasma.

The electrophoresis of proteoglycans and glycosaminoglycans on large-pore polyacrylamide gels which contained agarose as a support medium is described. The average pore diameter of the gels was sufficient to allow penetration of all but the very largest proteoglycans of articular cartilage, making it possible to distinguish “aggregates” from disaggregated molecules. The relative sizes of proteoglycans could be assessed, using only microgram quantities on these gels, as the mobilities were inversely related to hydrodynamic size as determined by gel chromatography. The method was able to separate single from double chains of chondroitin sulfate. On the other hand, the relative mobilities of separate chains of different types of glycosaminoglycans were a function of their charge density. The method has been applied to a pathological condition in which changes in the sizes of proteoglycans were evident.

THE guinea pig lymphocyte has long been known to contain a special inclusion body. Such lymphocytes, called Kurloff cells after their discoverer, increase greatly in number in pregnancy and were shown by Ledingham1 to be produced in both malesand females by administration of œstrogens. Marshall and Swettenham2, by histochemical methods, have shown that the inclusion body is composed of a mucoprotein–sulphated mucopolysaccharide complex. Further work on the chemistry of a mucopolysaccharide fraction by extraction of spleens of œstrogen-treated guinea pigs gave the following results